DISTINCT MECHANISMS CONTRIBUTE TO STRINGENT SUBSTRATE-SPECIFICITY OF TISSUE-TYPE PLASMINOGEN-ACTIVATOR

Tissue-type plasminogen activator (t-PA) has evolved to optimize cleav age of plasminogen (Pig) while minimizing cleavage of other potential protein and peptide substrates. We find that the S2 and S2' subsites o f t-PA are important determinants of specificity, and occupancy of the S3 subsite is essential for catalysis. t-PA efficiently hydrolyzes a protein substrate which incorporates an optimized substrate sequence, revealing the ability of the protease to participate in the highly sel ective cleavage of protein fusions. Surprisingly, t-PA cleaves this en gineered protein substrate with a K-m that is reduced 950-fold relativ e to the K-m for hydrolysis of the same target sequence within a pepti de. This reduction of K-m suggests that binding is facilitated by inte ractions between protein substrate and protease that are distant from the P4-P2' residues. We use this kinetic data to derive a model in whi ch several distinct mechanisms contribute to the remarkable specificit y of t-PA.